2 SUMMARY 1. Summary Cytomegaloviruses (CMVs), members of the β-herpesvirus family, infect between 60 to 90% of the human population throughout all geographic locations and socioeconomic groups. Acute CMV infection in humans and in mice is often clinically silent and is controlled by multiple arms of innate and adaptive immunity. However, complete clearance is never achieved, and similar to other herpesviruses, CMVs persist in their hosts lifelong in a latent form. Latent infection is believed to be a dynamic state, characterized by sporadic viral reactivation events which are readily controlled by the immune system. The importance of immune surveillance of CMV latency is evidenced by the severe consequences that CMV reactivation from latency causes in immunocompromised hosts. In particular, CD8 T cells are believed to play a major role by directly sensing virus reactivation events and terminating them by virtue of their effector functions. Evidence in support of this hypothesis is the exceptionally large CD8 T cell response that is induced in all seropositive humans as well as in feral mice, reaching up 30% of the total memory CD8 T cell pool. The mechanism leading to this impressive accumulation of memory CD8 T cells is largely unknown, but their highly activated phenotype is indicative of antigen-driven maintenance. The strong analogies between human CMVs (HCMVs) and murine (MCMVs) with respect to their interplay with the host immune systems make the mouse model a very valuable system to investigate the mechanism of antigen presentation to CD8 T cells during latency. Detailed analyses performed in C57BL/6 mice revealed that only a limited subset of CD8 T cells, referred to as inflationary CD8 T cells, contribute to this large response by increasing in number upon resolution of the acute infection. The majority of the MCMV-specific CD8 T cells that dominated the acute infection in fact contracted upon control of the infection and was present at low numbers with a T CM phenotype during latency. To investigate the molecular and cellular factors contributing to the development of two so diverse CD8 T cell response patterns within one host during MCMV infection, we have successfully generated MHC class I-restricted TCR transgenic mouse lines with specificity for the MCMV-derived epitope M38, representing an immunodominant epitope of the inflationary response. In addition, we are currently generating TCR transgenic mice specific for one epitope derived from the M45 protein of MCMV, which induces a conventional type of CD8 T cell response. 5

3 SUMMARY With the assumption that induction and maintenance of the inflationary response during MCMV latency is antigen dependent, we first aimed to identify the cell types responsible for antigen presentation to inflationary CD8 T cells. We approached this question by generating bone-marrow chimeras in which the M38 epitope could be presented on every cell type, or exclusively on hematopoietic cells, and normalized the initial population of M38-specific CD8 T cells by adoptively transferring M38-specific transgenic cells prior to infection. We found that antigen presentation on cells of non-hematopoietic origin is absolutely required for memory inflation, and that these cells are also the major reservoir of latent virus. In addition we observed that M38-specific CD8 T cells in the lymph nodes, which display predominantly a T CM phenotype, but not those in the periphery, where they are terminally differentiated, proliferated during latency in response to antigen presentation on non-hematopoietic cells. Based on these findings, we concluded that the inflationary pool in peripheral tissues is fueled by lymph-node resident CD8 T cells which are locally reactivated by MCMV antigens presented on non-hematopoietic cells, inducing their local expansion and migration to peripheral tissues where they can guarantee constant immunosurveillance. Our proposed model of antigen presentation to inflationary CD8 T cells during MCMV latency suggests that viral antigens are presented directly on infected non-hematopoietic cells. However it is not to exclude that DCs acquire viral antigens through phagocytosis of apoptotic infected cells, and present them to CD8 T cells in a process referred to as crosspresentation. We therefore aimed to assess the role of direct versus cross-presentation in the induction and maintenance of the CD8 T response during latency, and compared it to the responses induced during the acute phase of infection. Using of mouse model that selectively lacks the major subsets of cross-presenting DCs, we discovered that CD8 T cell inflation and priming rely on different antigen presentation pathways. While the first was largely independent on cross-presentation, confirming our original hypothesis of antigen sensing directly on latently infected non-hematopoietic cells, CD8 T cell priming was strongly reduced in the absence of cross-presenting DCs. This finding outlines a plausible mechanism of how the immune system could induce strong priming despite the numerous immune evasion strategies of CMV aimed at inhibiting antigen presentation to CD8 T cells. 6

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